Antagonists of ligands and uses thereof

ABSTRACT

The invention provides hetero-multivalent ligand binging agents (traps) for members of the TGF-β superfamily, as well as methods for making and using such constructs. In an embodiment of the invention there is provided a hetero-multivalent binding agent with affinity for a member of the TGF-β superfamily, said agent comprising the general structure (I): (&lt;bd1&gt;-linker1) k -[{&lt;bd1&gt;-linker2-&lt;bd2&gt;-linker3 r -} n -(&lt;bd3&gt;) m -(linker4-&lt;bd4&gt;) d ] h , where bd1, bd2, bd3 and bd4 are polypeptide binding domains having an affinity for different sites on the same member or for different members of the TGF-β superfamily, wherein at least two of bd1, bd2, bd3, and bd4 are different from each other.

FIELD OF INVENTION

The invention relates to the field of antagonists and, morespecifically, to polypeptide antagonists capable of use as single chainmultivalent ligand traps.

BACKGROUND OF INVENTION

Many undesirable biological processes occur via ligand binding to cellsurface receptors. Thus, it is sometimes desirable to have compounds andmethods to reduce or modulate such binding.

The TGF-β superfamily includes a number of ligands of biologicalsignificance.

TGF-β and Activin play critical pathogenic roles in many diseasesincluding the progression of cancer and uncontrolled fibrosis andscarring of tissues, e.g. kidney, lung and liver fibrotic diseases.Furthermore, Myostatin/GDF8 is another ligand which is related toActivin and which shares binding to the same Type II receptor(ActivinRIIb). Myostatin is a powerful inhibitor of skeletal musclegrowth and is a validated therapeutic target for muscle wasting diseasessuch as muscular dystrophy. Bone morphogenetic proteins (BMP), which areother ligands in the TGF-β family, have been implicated incardiovascular diseases. For example, high levels of both BMP2 and BMP4have been found in calcified atherosclerotic plaques and diseased aorticvalves.

Principal agents that target these ligands are ligand traps/antagoniststhat bind and sequester ligand. Two examples are: 1) anti-ligandantibodies and 2) soluble receptor ectodomains.

Efforts have been made to identify methods to reduce ligand binding bytrapping ligand and preventing its interaction with the cell surfacereceptors. Inhibition of certain ligands has been reported usinganti-ligand antibodies that trap and neutralize the ligand directly. Fortherapeutic and diagnostic applications, however, antibodies areproblematic, particularly due to issues arising from their large sizerestricting their ability to reach targets outside the bloodstream.

Soluble versions of receptor ectodomains antagonize ligands directly bybinding to them and preventing them from interacting with cell surfacereceptors. In the case of TGF-β, in animal models, expression of a TGF-βreceptor type II (TβRII) ectodomain (ED) partially restored hostimmunity and promoted tumor clearance, indicating that receptorectodomain-mediated neutralization of TGF-β inhibits tumor progression.It has been shown, however, that the efficacy of monovalent TβRII-ED toantagonize TGF-β is less than could be desired. Attempts to overcomethis led to the production of bivalent artificially dimerized forms ofversions of TβRII-ED, dimerized via fusion to either coiled-coil domainsor the Fc domain of IgG. This dimerization improved the antagonisteffect.

Bivalent receptor-based traps/neutralizers that antagonize multimericligand activity have the potential to act as therapeutic or diagnostic(imaging or non-imaging) agents for diseases/disorders caused byover-production/activity of the target ligand. It has been demonstratedthat non-covalent dimerization of TβRII-ED (for example, via fusion toheterodimerizing coil strands (coiled-coil TβRII-ED)), greatly enhancesthe antagonist potency of TβRII-ED (De Crescenzo et al., 2004, J. Biol,Chem. 279: 26013).

A significant disadvantage of the coiled-coil fused dimer is that thenon-covalent nature of the dimerization domain limits its potency, i.e.it dissociates at low concentrations such that a large portion of thecoil-fused receptor ectodomain will be acting as a monomer rather than adimer. Use of the Fc domain of IgG provides a covalent interaction, butat the cost of large size.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows amino-acid sequences corresponding to intrinsicallyunstructured regions in the extracellular portions of selectTGF-β-superfamily receptors. Residue numbering starts after signalpeptide.¹ SEQ ID 2 is present in TβRII and TβRIIb but at differentlocations, as indicated.

FIG. 1B shows amino-acid sequences corresponding to structuredligand-binding domain regions in the extracellular portions of selectTGF-β-superfamily receptors. Residue numbering starts after the signalpeptide.

FIG. 2A shows examples of sequences corresponding to natural linkers ofhetero-bivalent single-chain traps of the present invention resultingfrom fusion of the entire extracellular portions of selectTGF-β-superfamily receptors. Residue numbering corresponds to trapconstruct and starts after N-terminal tag. Fusion position is indicatedby (:).

FIG. 2B shows examples of sequences corresponding to embodiments ofartificial linkers for hetero-bivalent single-chain traps of the presentinvention at varying sequence identity to natural linker sequences.Residue numbering corresponds to single-chain trap. Changed amino-acidresidues relative to natural sequence are underlined. *This linkercorresponds to the (TbR-II)2 referred to in the text.

FIG. 2C shows examples of sequences corresponding to varying the linkerlength for embodiments of hetero-bivalent single-chain traps of thepresent invention by deleting or repeating of natural sequences, or byinserting of artificial sequences, into the natural linker sequence.Residue numbering corresponds to trap construct and starts afterN-terminal tag. Added amino-acid sequences, either natural orartificial, are underlined. Deletions are denoted by dashes. Naturallinker sequences are also included as reference.

FIG. 3 shows a graphical depiction of inhibition of TGF-β3 (A) andTGF-β2 (B) signaling in Mv1Lu luciferase reporter cells by an embodimentof homo-bivalent traps (TβRII)² and (TβRIIb)² compared to TβRII-Fc,TβRII-ED monomer and pan-specific TGF-β neutralizing antibody 1D11. Thehomo-bivalent TGF-β traps efficiently neutralize TGF-β3 but do notneutralize TGF-β2.

FIG. 4A provides schematic diagrams exemplifying embodiments of in-linefusions of receptor ectodomains leading to embodiments of hetero-valentsingle-chain traps of TGF-β-superfamily growth factors.

FIG. 4B shows amino-acid sequences exemplifying embodiments ofhetero-valent single-chain traps (ligand binding agents) ofTGF-β-superfamily growth factors, corresponding to the domainorganization diagrams depicted in FIG. 4A. underlined: natural linker orsequence; underlined-italics: artificial linker; bold-Italics: TbR-I-EDstructured domain; bold: TbR-II-ED structured domain; regular:unstructured region of TbR-II-ED that becomes structured in the ternarycomplex TfβR-I/TβR-II/TGF-β [Groppe at al. 2008].

FIG. 5 shows amino-acid sequences exemplifying embodiments ofhetero-bivalent single-chain traps of TGF-β-superfamily growth factorsusing natural linkers of varying length and composition. underlined:natural linker or sequence; bold-italics: TbR-I-ED structured domain inTbR-I/II traps and ActR-IIa structured domain in ActR-IIa/BMPR-Ia traps;bold: TbR-II-ED structured domain in TbR-I/II traps and BMPR-Iastructured domain in ActR-IIa/BMPR-Ia traps; regular: unstructuredregion of TbR-II-ED that becomes structured in the ternary complexTbR-I/TbR-II/TGF-b [Groppe et al. 2008].

FIG. 6 shows amino-acid sequences exemplifying embodiments ofhetero-bivalent single-chain traps of TGF-β-superfamily growth factorsusing artificial linkers of varying length and composition, underlined:natural linker or sequence; underlined-italics: artificial linker;bold-italics: TbR-I-ED structured domain in TbR-I/II traps and ActR-IIastructured domain in ActR-IIa/BMPR-Ia traps; bold: TbR-II-ED structureddomain in TbR-I/II traps and BMPR-Ia structured domain inActR-IIa/BMPR-Ia traps; regular: unstructured region of TbR-II-ED thatbecomes structured in the ternary complex TbR-I/TbR-II/TGF-b [Groppe etal. 2008].

FIG. 7 shows images of feasibility studies of embodiments ofhetero-bivalent trap constructs with natural linkers fromthree-dimensional structural models. Shown are molecular mechanicsenergy-minimized natural linkers for TβR-I/II-v1 and ActR-IIa/BMPR-Ia-v1hetero-bivalent single-chain traps in complex with the TGF-β3 and BMP-2growth factors, respectively. Each growth factor covalent dimer isrendered in gray. Each single-chain trap is rendered in black, andconsists of two distinct folded binding domains and an interveningunstructured linker. Each dot indicates the point of fusion in thelinker region between two distinct receptor ectodomains to generate thesingle-chain trap. Arrowheads indicate polypeptide chain direction inthe trap's linker. Two 90°-rotated views are provided for each complex.See FIG. 5 for amino acid sequences in the structured binding domainsand the intervening linker.

FIGS. 8 A and B are graphical depictions of neutralization of TGF-β1 (A)and TGF-β2 (B) by hetero-valent TβR-I/II-v1 and TβR-I/II-v2 traps. 293cells were transfected with TβR-I/II-v1 or TβR-I/II-v2. Conditionedmedia (CM) was collected after 2 days. Mv1 Lu TGF-β luciferase-reportercells were treated with 20 pM TGF-β plus various dilutions of CM andthen analyzed for their luciferase levels (i.e. TGF-β-induced luciferaseresponse). The bars show the average response levels, relative to thecontrol CM, for each dilution (error bars=SEM for triplicate samples).

FIG. 9 shows TGF-β1 (A) and TGF-β2 (B) neutralization curves forpurified single-chain TβR-I/II-v1 trap protein.

a graphical depiction of inhibition of TGF-β1 (A) and TGF-β2 (B)signaling in Mv1Lu luciferase reporter cells by an embodiment ofpurified hetero-bivalent trap TβR-I/II-v1 (RLU=relative luciferaseunits).

SUMMARY OF INVENTION

The invention relates to ligand binding agents capable of permittingmodulation of cellular response to members of the TGF-β superfamily bybinding one or more members of the TGF-β superfamily and preventinginteraction with cellular receptors, and methods of designing and usingsuch agents. The ligand binding agents taught herein are preferablysingle chain multivalent ligand binding agents. However, it would bepossible to link such single-chain constructs to other uni- ormultivalent molecules and/or to combine two or more such single chaintraps using multimerization domains known in the art (e.g. coiled-coildomains, Fc domains, pentabodies) to form a multimeric trap if sodesired and any such trap having a multivalent single chain portionfalls within the scope of the present invention.

In an embodiment of the invention there are provided methods andprocesses to engineer hetero-multivalent receptor ectodomains using asingle-chain approach.

The ligand binding agents of the invention are preferablyhetero-multivalent ligand traps, having at least two binding domains(bd) which recognize different sites on (or the same site of differentportions of) the same member of the TGF-β superfamily. The bindingdomains may be modified, for example to facilitate purification, so longas such modifications do not reduce binding affinity to unacceptablelevels.

In an embodiment of the invention there are provided hetero-multivalentligand traps having the general Structure I:

(<bd1>-linker1)_(k)-[{<bd1>-linker2-<bd2>-linker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h),

where:

-   -   n and h are independently greater than or equal to 1;    -   d, f, m and k are independently equal to or greater than zero;    -   bd1, bd2, bd3 and bd4 are polypeptide binding domains        independently having an affinity for a member of the TGF-β        superfamily, wherein at least two of bd1, bd2, bd3, and bd4 are        different from each other; such that the interface of        complementary interactions with TGF-beta isoforms is increased;        and,    -   linker1, linker2, linker3 and linker4 are unstructured        polypeptide sequences; wherein the number of amino acids in each        linker is determined independently and is greater than or equal        to X/2.5; where, X equals the shortest linear distance between:        -   a) the C-terminus of an isolated form of the binding domain            that is located at the N-terminus of the linker and that is            specifically bound to its ligand; and,        -   b) the N-terminus of an isolated form of the binding domain            that is located at the C-terminus of the linker and that is            specifically bound to its ligand.

Subject to the constraints described herein, linkers 1, 2, 3, and 4 maybe the same or different. In certain embodiments the linker is between 6and 60 amino acids in length

Also provided are nucleic acid sequences encoding such ligand traps.

In certain embodiments of the invention, the member of the TGF-βsuperfamily to which the binding domains (bd) have affinity is selectedfrom the group consisting of: TGF-β1, TGF-β2, TGF-β3, activin βA,activin βB, activin βC, activin βE, bone morphogenic protein (BMP) 2,BMP 3, BMP4, BMP 5, BMP 6, BMP 7, BMP 8, BMP 9, BMP 10, BMP 11, BMP 12,BMP 13, BMP 14, BMP 15, growth differentiation factor (GDF) 1, GDF 3,GDF 8, GDF 9, GDF 15, Nodal, Inhibin α, anti-Mullerian Hormone, Lefty 1,Lefty 2, arteman, Persephin and Neurturin.

In an embodiment, one or more of bd1, bd2, bd3, and bd4 may be selectedfrom SEQ ID NO 12-17.

In an embodiment of the invention the binding agent comprises one ormore of SEQ ID NOs 44-64.

In an embodiment of the invention the binding agent comprises one ormore of SEQ ID NO 1-11 or 18-43, 65-107, or an underlined region of oneor more of SEQ ID NO 44-64 as they appear in FIGS. 4, 5 and or 6 as alinker sequence.

In an embodiment of the invention there are provided polypeptidesequences useful in binding TGF-β. In some instances, such sequences areSEQ ID NOs 44, 45 and/or 55 and/or variants thereof.

In an embodiment of the invention, there are provided heterovalent TGF-βbinding agents and methods for their use in modulating the response of acell to a member of the TGF-β superfamily such as TGF-β1 and/or TGF-β2.

In an embodiment of the invention there are provided methods and uses ofmodelling of molecular mechanics of unstructured polypeptide sequencesfunctioning as linkers between two binding domains having affinity fordifferent sites on a member of the TGF-β superfamily.

The invention also provides a method of designing a hetero-multivalentbinding agent useful in modulating responsiveness of a cell to a memberof the TGF-β superfamily, said method comprising:

a) identifying a member of the TGF-β superfamily of interest;b) obtaining at least two different polypeptide binding domains havingaffinity for different sites on the same member or for different membersof the TGF-β superfamily;c) obtaining an unstructured polypeptide linker of at least a number ofamino acids equal to (X/2.5) where X equals the shortest linear distancebetween:

-   -   (i) the C-terminus of an isolated form of the binding domain        that is located at the N-terminus of the linker and that is        specifically bound to its ligand; and,    -   (ii) the N-terminus of an isolated form of the binding domain        that is located at the C-terminus of the linker and that is        specifically bound to its ligand; and,        d) modelling the linker between the binding domains and carrying        out molecular mechanics and/or dynamics simulations to        substantially minimize the interaction energy and reduce steno        and electrostatic incompatibility between the linker and the        member of the TGF-β superfamily.

The design method can optionally be expanded to further include a stepe) of producing a fusion protein comprising the two polypeptide bindingdomains joined by the unstructured polypeptide linker.

The ligand binding agents disclosed herein are also useful inpurification of ligand, for example, by immobilization on an inertmatrix on a solid support, for example, on nanoparticles to concentratelevels of ligand in a sample.

The invention also provides novel polypeptide sequences useful in avariety of applications. These sequences include SEQ ID NOs 44 to 64.Also provided are nucleic acid sequences encoding these polypeptidesequences.

Also provided is a method of modulating the response of a cell to TGF-βin its environment, said method comprising exposing the cell to amultivalent ligand trap comprising a ligand binding agent (ligand trap)disclosed herein.

In an embodiment of the invention there is provided a binding agenthaving the general structure V.

wherein R₁, R₂, R₃/R₄, R₅, R₆, R₇, R₈, R₉, may be the same or different,may not be present and when present, may independently be one or more ofa protein for targeting, e.g. a single domain antibody, a radiotherapyagent, an imaging agent, a fluourescent dye, a fluorescent protein tag,a cytotoxic agent for chemotherapy a nano particle-based carrier, apolymer-conjugated to drug, nanocarrier or imaging agent, a stabilizingagent, a drug a nanocarrier, a dendrimer and a support for use inpurification or concentration of ligand; and wherein bd1, bd2, bd3, bd4,linker1, linker2, linker3, linker4, k, f, n, m, d, and h are defined asin Structure I. In light of the disclosure herein, one skilled in theart can select suitable R-groups for diagnostic therapeutic or otherapplications.

In an embodiment of the invention there is provided a polypeptidecomprising a region having at least 80%, 85%, 90%, 95%, 98%, 99%sequence identity to one or more of SEQ ID NOs 44-64. In some instancesthis polypeptide has a region with at least 90%, 95%, 98%, 99% sequenceidentity to one or more of SEQ ID NOs 44-64.

In an embodiment of the invention there is provided a nucleic acidsequence encoding a polypeptide disclosed herein.

In an embodiment of the invention there is provided a method ofmodulating the response of a cell to a TGF-β superfamily member in itsenvironment, said method comprising exposing the cell to a ligandbinding agent disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the field of antagonists and, morespecifically, to polypeptide antagonists capable of use as single chainmultivalent ligand traps.

The present invention provides a single-chain non-naturally occurringpolypeptide useful as a ligand binding agent. The ligand binding agentcomprises structured ligand-binding domains (denoted bd) derived from orbased on the extracellular portion of a natural receptor or receptors,joined by one or more polypeptide linkers. The ligand binding agentprovides a multivalent binding agent and does not require fusion to anyconventional dimerizing or multimerizing moieties such as coiled-coildomains of Fc domains in order to be multivalent.

In one aspect of the present invention, there is provided ahetero-multivalent binding agent with affinity for one or more than onemember of the TGF-β superfamily, said agent comprising the generalStructure I:

(<bd1>-linker1)_(k)-[{<bd1>-linker2-<bd2>-linker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h),

where:

-   -   n and h are independently greater than or equal to 1;    -   d, f, m and k are independently equal to or greater than zero;    -   bd1, bd2, bd3 and bd4 are polypeptide binding domains        independently having an affinity for a member of the TGF-β        superfamily, wherein at least two of bd1, bd2, bd3, and bd4 are        different from each other; such that the interface of        complementary interactions with TGF-beta isoforms is increased;        and,    -   linker1, linker2, linker3 and linker4 are unstructured        polypeptide sequences, wherein the number of amino acids in each        linker is determined independently and is greater than or equal        to X/2.5, where X equals the shortest linear distance between:        -   (a) the C-terminus of an isolated form of the binding domain            that is located at the N-terminus of the linker and that is            specifically bound to its ligand; and,        -   (b) the N-terminus of an isolated form of the binding domain            that is located at the C-terminus of the linker and that is            specifically bound to its ligand.

Depending on the values selected for d, f, h, k, m, and n, the ligandtrap structure may comprise a large number of repeating units in variouscombinations or may be a relatively simple structure such as StructureII <bd1>-linker-<bd2>.

The ligand-binding agents, also referred to herein as “traps” or “ligandtraps”, of the present invention are multivalent as they comprisemultiple binding domains (bd). The term “multivalent” includes bivalent(2 bd), trivalent (3 bd), quadruvalent (4 bd), and greater numbers ofbinding domains. The multivalent binding agents are heterologous(“hetero-”), as at least two binding domains are different from eachother and recognize different sites on the same member of the TGF-βsuperfamily. or recognize different members of the TGF-β superfamily.

The hetero-multivalent binding agents of the present invention may haveaffinity for one or more than one member of the TGF-β superfamily. Bythe term “affinity”, it is meant the free energy of the process ofbinding between the said molecules.

The term “TGF-β superfamily” refers to the family of structurallyrelated cell regulatory proteins, of which TGF-β is a founding member.These proteins are only active as homo- or heterodimer, the two chainsbeing linked by a single disulfide bond. Members of the TGF-βsuperfamily to which the binding domains (bd) have affinity may include,but are not limited to TGF-β1, TGF-β2, TGF-β3, activin βA, activin βB,activin βC, activin βE, bone morphogenic protein (BMP) 2, BMP 3, BMP4,BMP 5, BMP 6, BMP 7, BMP 8, BMP 9, BMP 10, BMP 11, BMP 12, BMP 13, BMP14, BMP 15, growth differentiation factor (GDF) 1, GDF 3, GDF 8, GDF 9,GDF 15, Nodal, Inhibin α, anti-Mullerian Hormone, Lefty 1, Lefty 2,arteman, Persephin and Neurturin.

The binding domains in the ligand traps of the present invention maycomprise any suitable polypeptide that has affinity for a member of theTGF-β superfamily. The binding domains within a hetero-multivalent trapof the present invention are independent of each other, and as such, thebinding domains may have different affinities. Each binding domainregion of the single-chain polypeptide may be selected for its abilityto bind a growth-factor ligand having a covalently-stabilized dimericquaternary structure; each binding domain may have affinity to one ormore member of the TGF-β superfamily. The bd may be a receptor for agrowth factor selected from within the TGF-β family, e.g., but notlimited to transforming growth factor beta (TGF-β), bone morphogeneticprotein (BMP), activin, myostatin, and including their naturallyoccurring isoforms.

In one example, the polypeptide binding domains may be designed based onthe extracellular portion of the cognate natural receptors of the growthfactors of the TGF-β superfamily. In a further example, the naturalreceptors from which the polypeptide binding domain is designed may be,but is not limited to TβR-I-ED, TβR-II-ED, ActR-IIa-ED, or BMPR-Ia-ED,or any other natural receptor ectodomain. In yet another non-limitingexample, the binding domains may be selected from SEQ ID NOs: 12-17. Asused herein “an isolated form” of a binding domain is a form of thatbinding domain able to act as a monovalent monomer.

The binding domains may be modified, for example to facilitatepurification, so long as such modifications do not reduce bindingaffinity to unacceptable levels.

Within a hetero-multivalent ligand trap of the present invention, thebinding domains that differ from each other will bind different sites onthe one or more member of the TGF-β superfamily. In a non-limitingexample, in a hetero-bivalent ligand trap, the binding domains may bindto distinct sites on each member of the TGF-β superfamily; however, thehetero-bivalent ligand trap may bind a single member of the TGF-βsuperfamily at any given time.

The binding domains (bd) of the ligand traps may be joined by a flexiblepolypeptide linker region. The linkers (1, 2, 3, and 4) in the traps ofthe present invention may be the same or different. The linker regionprovides a segment that is distinct from the structured ligand bindingdomains and thus can be used for conjugation to accessory molecules (forexample, molecules useful in increasing stability such as PEGylationmoieties) or cargo molecules such as contrast agents (for imaging)without having to chemically modify the binding domains. The linker mayinclude an unstructured amino acid sequence that may be either the sameas or derived from conservative modifications to the sequence of anatural unstructured region in the extracellular portion of the receptorfor the ligand of interest or another receptor in the TGF-β superfamily.In other instances, such linkers may be entirely artificial incomposition and origin but will contain amino acids selected to providean unstructured flexible linker with a low likelihood of encounteringelectrostatic or steric hindrance complications when brought into closeproximity to the ligand of interest.

The length of the linker is considered to be the number of amino acidsbetween:

-   -   (a) the C-terminal main chain carbon atom of the binding domain        located at the linker's N-terminal end; and    -   (b) the N-terminal main-chain nitrogen atom of binding domain        located at the linker's C-terminal end.

Linker length will be considered acceptable when it permits bindingdomains located on each of the N- and C-termini of the linker to bindtheir natural binding sites on their natural ligand such that, with bothbinding domains so bound, the ligand is bound with a higher affinitythan it would be bound by binding of only one of the binding domains.

In some instances, the number of amino acid residues in the linker ofeither natural or artificial origin is selected to be equal to orgreater than the minimum required distance for simultaneous (bridged)binding to two binding sites on the target growth factor. A non-limitingexample of such a determination is given in the section “Feasibilityassessment procedure for designed single-chain bivalent traps”. Examplesof natural and artificial linker sequences of varying length are givenin FIG. 2B, FIG. 2C, Table 2, FIG. 5 and FIG. 6. For example, andwithout wishing to be limiting in any manner, the linker length may bebetween about 18-80 amino acids, 25-60 amino acids, 35-45 amino acids,or any other suitable length.

In one example of the invention there is provided ligand binding agentswherein the intervening linker sequence is composed of native aminoacids, the sequence of which is based on the receptor ectodomains (e.g.the various linkers shown in FIG. 2A and the “repeat” and “delete”linkers shown in FIG. 2C) or conservative substitutions of natural orunnatural amino acids into such regions, or reversal of such natural ormodified sequences. It will frequently be considered preferable to useunstructured regions from these receptor ectodomains as the template forlinker design. Once linkers have been designed, it will generally bepreferred to test their effectiveness using the procedures describedherein or other substantially functionally equivalent procedures.Routine testing for immunogenicity may be desired for in vivo use.

Non-limiting examples of useful linkers may be found in the amino acidsequences in SEQ ID NOs 1-11 and 18-43 which should be readconventionally with the N-terminus on the left and the C-terminus on theright, and in corresponding reverse sequences having the same aminoacids but wherein the C-terminus is on the left and the N-terminus is onthe right as the sequences are written in full. In some embodiments,such reverse sequences may be produced using D-amino acids. Whereimmunogencity is of concern, it may be desired to screen such reversesequences for immunogenicity at an early stage (For examples of reversesequences, see SEQ ID NOs: 65-107). Amino acids sequences in the presentdocument are written N-terminus to C-terminus, unless otherwise noted.All sequences disclosed herein (except SEQ ID NO: 65-107) are disclosedas using L-amino acids; the use of a D-amino acid is considered avariant affecting the percent sequence identity to the sequences asstated.

In some instances, the linker may be independently selected to havevarying degrees of sequence identity to naturally occurring unstructuredamino acid sequences found in the native receptor sequence in theregions flanking the ligand binding domain, for example 70%, 80%, 90%,95%, 98%, 99% or 100% sequence identity, whereas for entirely artificiallinkers (e.g. poly-Gly or poly-Ser linkers), sequence identity will beeven lower. Examples of linker sequences of varying degree of identityto the natural receptor sequence are shown in FIG. 2B, FIG. 5, and Table2.

In addition to linkers disclosed elsewhere herein, the polypeptidesequences of Table 2 may be useful as linkers or components thereof.These polypeptides may be useful when produced using either L- orD-amino acids. However, with respect to SEQ ID NOs 65 to 107 use ofD-amino acids will frequently be preferred.

TABLE 2 Non-limiting examples of linkers. SEQ ID Linker Sequence NO:COOH-IPPHVQKSVNNDMIVTDNNGAVKFP-NH2 65 COOH-SEEYNTSNPD-NH2 66 COOH- 67IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFP-NH2 COOH-AALLPGAT-NH268 COOH-PTTVKSSPGLGPVE-NH2 69 COOH-AILGRSE-NH2 70COOH-EMEVTQPTSNPVTPKPPYYNI-NH2 71 COOH-SGRGEAET-NH2 72COOH-EAGGPEVTYEPPPTAPT-NH2 73 COOH-QNLDSMLHGTGMKSDSDQKKSENGVTLAPED-NH274 COOH-PVVIGPFFDGSIR-NH2 75 COOH- 76QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF-NH2 COOH- 77QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF-NH2 COOH- 78ALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIEL-NH2 COOH- 79TQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP-NH2 COOH- 80RECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEP PPTAPT-NH2 COOH- 81TLPFLKCYCSGHCPDDAINNTCITNGHCFAIIEEDDQGETTLASGCMKYEGSDFQCKDSPKAQLRRTIECCRTNLCNQYLQPTLPPVVIGPFFDGSIR-NH2COOH-SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP-NH2 82 COOH- 83SEEYNTSNPDIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNN GAVKFP-NH2COOH-EAGGPEVTYEPPPTAPTSGRGEAET-NH2 SEQ ID NO 50 84COOH-PVVIGPFFDGSIRQNLDSMLHGTGMKSDSDQKKSENGVTLAPED-NH2 85COOH-PVVIGPFFDGSIRGNLDSMLHGTGMKSDSDQKKSENGVTLAPED-NH2 86COOH-SEEYNTSNPDGPPHVQKSVNNDMIVTDNNGAVKFP-NH2 87COOH-EAGGPEVTGEPPPTAPTSGRGEAET-NH2 88 COOH- 89SEEYNTSNPDGGRHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDN NGAVKFP-NH2COOH-SEEYNTSNPDGGPHVQKSVNNDMIVTDNNGAVKFP-NH2 90COOH-SEEYNTSNPDGGRHVQKSVNNDMIVTDNNGAVKFP-NH2- 91COOH-SEEYNTSNPSGGGSGGGSGGGMEAQKDEIICPSCNRTAHPLRHINND 92MIVTDNNGAVKFP-NH2 COOH-SEEYNTSNPSGGGSGGKSVNNDMIVTDNNGAVKFP-NH2 93COOH-SEEYNTSNPSGGGSGGGSGGGDMIVTDNNGAVKFP-NH2 94 COOH- 95SEEYNTSNPDIPPHVQKSGGGSGGGSGGGSGGGSGGGSGGGSGGNNDMIV TDNNGAVKFP-NH2 COOH-96 SEEYNTSNPDGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGNNDM IVTDNNGAVKFP-NH2COOH-SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP-NH2 97 COOH- 98SEEYNTSNPDIPPHVQKSVNNDMIPPHVQKSVNNDMIVIDNNGAVKFP-NH2COOH-SEEYNTSNPPHVQKSVNNDMIVTDNNGAVKFP-NH2 99COOH-SEEYNTSNPDGGGGGGGGIPPHVQKSVNNDMIVIDNNGAVKFP-NH2 100 COOH- 101SEEYNTSNPDGGGSGGGSGGGSIPPHVQKSVNNDMIVTDNNGAVKFP-NH2 COOH- 102SEEYNTSNPDIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNN GAVKFP-NH2 COOH-103 SEEYNTSNPDIPPHVQKSDVEMEAQKDERTAHPLRHINNDMIVTDNNGAVKFP-NH2COOH-EAGGPEVTYEPPPTAPTSGRGEAET-NH2 104COOH-EAGGPEVTYEPPPTAPTGGGGGGGGGGSGRGEAET-NH2 105COOH-PVVIGPFFDGSIRQNLDSMLHGTGMKSDSDQKKSENGVTLAPED-NH2 106COOH-PVVIGPDGSIRQNLDSHGTGMKSDSDQKKSENGVTLAPED-NH₂ 107 Also contemplatedare nucleic acid sequences encoding such linkers.

In some instances, it may be desirable to subject the polypeptide-basedlinking design of the ligand binding agents disclosed herein tooptimization of characteristics desired for a particular application.For example, the linker may be modified in length and composition basedon atomic-level simulations and knowledge-based design in order toimprove binding affinity, specificity, immunogenicity and stability.This is applicable to a wide range of molecular systems exhibitinghomomeric, heteromeric, dimeric and multimeric ligand-receptorstructural characteristics. Additional different binding domains can beincorporated to generate multivalent traps with even higher bindingpotency.

Linkers may be designed to facilitate purification of the linker and/orligand binding agent. The exact purification scheme chosen willdetermine what modifications are needed, for example and without wishingto be limiting, additions of purification “tags” such as His tags iscontemplated; in other examples, the linker may include regions tofacilitate the addition of cargo or accessory molecules. When suchadditions affect the unstructured nature of the linker or introducepotential electrostatic or steric concerns, appropriate increases to thelinker length will be made to ensure that the two binding domains areable to bind their respective sites on the ligand. In light of themethods and teachings herein, such determinations could be maderoutinely by one skilled in the art.

In an embodiment of the invention in which the ligand-binding domainsand the linker contain primarily natural sequences they would notordinarily be expected to be severely immunogenic or toxic in a typicalpatient.

The ligand binding agents of the present invention may be provided assingle-chain polypeptide molecules. The fusion proteins may comprise thesequence (excluding the signal peptide) of the natural extracellularportion of one receptor repeated one or more times and the sequence(excluding the signal peptide) of the natural extracellular portion ofanother receptor repeated one or more times. Constructs may be providedwith two or more structured domains for binding to selectTGF-β-superfamily ligand(s), spaced by unstructured flexible linker(s)formed by fusing the unstructured C-terminus of one domain to theunstructured N-terminus of another domain. The natural linkers may alsoprogressively be substituted by artificial sequences, as well as variedin length

In a non-limiting example, the binding agent may comprise one or more ofSEQ ID NOs: 44-64, or sequences substantially identical thereto. In aspecific, non-limiting example, there is provided polypeptide sequencesuseful in binding TGF-β. In some instances, such sequences are SEQ IDNOs 44, 45 and/or 55, sequences substantially identical thereto, and/orvariants thereof. A substantially identical peptide may comprise one ormore conservative amino acid mutations. It is known in the art that oneor more conservative amino acid mutations to a reference peptide mayyield a mutant peptide with no substantial change in physiological,chemical, or functional properties compared to the reference peptide; insuch a case, the reference and mutant peptides would be considered“substantially identical” polypeptides. Conservative amino acid mutationmay include addition, deletion, or substitution of an amino acid; aconservative amino acid substitution is defined herein as thesubstitution of an amino acid residue for another amino acid residuewith similar chemical properties (e.g. size, charge, or polarity).

In a non-limiting example, a conservative mutation may be an amino acidsubstitution. Such a conservative amino acid substitution may substitutea basic, neutral, hydrophobic, or acidic amino acid for another of thesame group. By the term “basic amino acid” it is meant hydrophilic aminoacids having a side chain pKa value of greater than 7, which aretypically positively charged at physiological pH. Basic amino acidsinclude histidine (His or H), arginine (Arg or R), and lysine (Lys orK). By the term “neutral amino acid” (also “polar amino acid”), it ismeant hydrophilic amino acids having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Polar amino acids include serine (Ser or S), threonine (Thror T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N),and glutamine (Gin or Q). The term “hydrophobic amino acid” (also“non-polar amino acid”) is meant to include amino acids exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg (1984). Hydrophobic aminoacids include proline (Pro or P), isoleucine (Ile or I), phenylalanine(Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp orW), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).“Acidic amino acid” refers to hydrophilic amino acids having a sidechain pKa value of less than 7, which are typically negatively chargedat physiological pH. Acidic amino acids include glutamate (Glu or E),and aspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences;it is determined by calculating the percent of residues that are thesame when the two sequences are aligned for maximum correspondencebetween residue positions. Any known method may be used to calculatesequence identity; for example, computer software is available tocalculate sequence identity. Without wishing to be limiting, sequenceidentity can be calculated by software such as BLAST-P, Blast-N, orFASTA-N, or any other appropriate software that is known in the art. Thesubstantially identical sequences of the present invention may be atleast 80% identical; in another example, the substantially identicalsequences may be at least 80, 85, 90, 95, or 100% identical at the aminoacid level to sequences described herein.

In another aspect, the ligand binding agent of the present invention maycomprise the general Structure II:

<bd1>-linker2-<bd2>.

In yet another aspect of the present invention, the ligand binding agentcomprises the general Structure III

<bd1>-(linker2-<bd2>)_(n).

Another aspect of the invention provides a ligand trap comprising thegeneral Structure IV:

([bd1]-[linker1]-[bd1])_(f)-[linker2]-([bd2]-[linker3]-[bd3])_(g),

where f and g are greater than or equal to one.

In an embodiment where bd2 and bd3 are the same, and f and g are thesame number, this can result in a substantially mirror symmetricstructure around linker 2, subject to differences in the linkers. Ininstances where bd3 is different from bd2 and/or where f and g aredifferent numbers, different structures will be produced. It is withinthe capacity of one of ordinary skill in the art to select suitablebinding domains, linkers, and repeat frequencies in light of thedisclosure herein.

In an embodiment of the invention, a non-naturally occurringsingle-chain hetero-bivalent polypeptide is produced by the inlinefusion of two or more different structured ligand-binding domains(denoted <bd1>, <bd2>, <bd3> and <bd4>) from the extracellular portionof distinct natural receptors, and which is not fused to any dimerizingor multimerizing moieties. In some instances, this polypeptide may havethe general structure <bd1>-linker2-<bd2>. In some instances, thebinding domains may be selected from the ectodomains of the TβR-II andTβRI receptors, and fused to produce hetero-bivalent single-chain trapsactive against TGF-β isoforms. In other instances, the binding domainsmay be selected from the ectodomains of the ActR-IIa and BMPR-Iareceptors and fused to generate single-chain hetero-bivalent trapsactive against activin, myostatin and BMP isoforms. In otherembodiments, the binding domains are selected from other receptors tomembers of the TGF-β superfamily.

In another embodiment of the invention a non-naturally occurringsingle-chain hetero-trivalent polypeptide is produced by the inlinefusion of two or more different structured ligand-binding domains(denoted bd1 and bd2) from the extracellular portion of distinct naturalreceptors, and which is not fused to any dimerizing or multimerizingmoieties. In some instances, this polypeptide may have the generalstructure [bd1]-linker1-[bd2]-linker2-[bd2]. In other instances, thispolypeptide may have the general structure[bd1]-linker1-[bd1]-linker2-[bd2]. In some instances, [bd1] and [bd2]may be selected from the ectodomains of the TβR-II and TβRI receptors,and fused to produce hetero-bivalent single-chain traps active againstTGF-β isoforms. In other instances, bd1 and bd2 may be selected from theectodomains of the ActR-IIa and BMPR-Ia receptors and fused to generatesingle-chain hetero-bivalent traps active against activin, myostatin andBMP isoforms.

In another embodiment of the invention a non-naturally occurringsingle-chain hetero-tetravalent polypeptide is produced by the inlinefusion of two or more identical or different structured ligand-bindingdomains from the extracellular portion of natural receptors repeatedtwice or more times in various orders. In an embodiment to the inventionthis hetero-tetravalent polypeptide is not fused to any dimerizing ormultimerizing moieties. In one embodiment, this polypeptide may have thegeneral structure [bd 1]-linker1-[bd2]-linker2-[bd1]-linker1-[bd2]. Inother instances, this polypeptide may have the general structure[bd1]-linker1-[bd1]-linker2-[bd2]-linker3-[bd2]. In one embodiment, thispolypeptide may have the general structure[bd1]-linker1-[bd2]-linker2-[bd2]-linker3-[bd1]. In some instances,[bd1] and [bd2] may be selected from the ectodomains of the TβR-II andTβR-I receptors, and fused to produce single-chain hetero-tetravalenttraps active against TGF-β isoforms. In other instances, [bd1] and [bd2]may be selected from the ectodomains of the ActR-IIa and BMPR-Iareceptors and fused to generate single-chain hetero-tetravalent trapsactive against activin, myostatin and BMP isoforms.

Specific non-limiting examples of heteromeric single-chain traps againstTGF-β, and in accordance with the present invention, are representedschematically as well as with full sequence details in FIGS. 4A and 4B.FIGS. 5 and 6 provide additional examples of hetero-bivalent trapsagainst TGF-beta and BMP based on the crystal structures of respectiveternary complexes. Natural linkages between different binding domainsare found in the traps listed in FIG. 5, while artificial linkages intraps are shown in FIG. 6. Molecular models of two hetero-bivalent trapswith natural linkers are given in FIG. 7, one against TGF-β(TβR-II/I-v1) and one against BMP (ActR-IIa/BMPR-Ia-v1).

The overall molecular mass of bivalent ligand binding agents disclosedherein before glycosylation is between about 26 kDa and 37 kDa, and theoverall mass following typical glycosylation is between about 35 kDa and60 kDa. Many of the binding agents taught herein will have a lowermolecular mass compared with competing multivalent receptor-basedneutralizing agents or comparable multimeric ligand traps constructedusing known multimerization domains.

TABLE 1 Example of Selected Ligand Trap Sizes Actual (withglycosylation) Agent Predicted for protein based on SDS-PAGE (TβRII)² 34kDa 50-60 kDa (TβRIIb)² 37 kDa 50-60 kDa (ActRIIB)² 30 kDa 50-60 kDa(BMPR1a)² 29 kDa 40-50 kDa RIIEcoil + RIIKcoil 37 Kd + 40 kDa = 77 kDa TβRII-Fc 60 Kd + 60 kDa = 120 kDa TβR-I/II-v1 26 kDa 35-45 kDa

The multivalent polypeptide ligand binding agents described herein allowfor high affinity and specificity by single-chain multivalency. Thissingle-chain attribute is fundamentally different from existingmulti-chain agents such as Fc-based fusions (covalent dimer),E/K-coiled-coil-based fusions (non-covalent dimer), or describedcytokines and ligand traps that include fused multimerizing moieties.Additionally, the hetero-bivalent ligand traps of the present inventionhave clear advantages over the molecules described in published PCTapplication WO 2008/113185 (O'Connor-McCourt et al). The traps ofO'Connor-McCourt et at show limitation of trap affinity in some cases,and are not able to neutralize multiple TGF-β isoforms.

Without wishing to be bound by theory, TpRI/RII hetero-bivalent trapsshow improved binding affinity relative to either monovalent TβRII-ED orTβRI-ED traps alone due to an increase in the interface of complementaryinteractions with TGF-β isoforms. Pan-specific neutralization of TGF-β1,-β2, -β3 by TβRI/RII hetero-bivalent traps relative to monovalent trapsis also due to this increase in affinity. That is, although the TβRI/RIIhetero-bivalent traps may still bind TGF-β1 and TGF-β3 with higheraffinity than TGF-β2, affinities to all TGF-β isoforms may be increased,including the TGF-β2 isoform. In the case of homo-bivalent traps like(TβRII)², the increase in affinity due to avidity does not materializeinto pan-specificity because of three amino acid differences betweenTGF-β2 and the other isoforms that impair its high-affinity binding toTβRII (De Crescenzo et al. 2006, J. Mol. Biol. 355:47, Baardsnes et al.2009, Biochemistry 48: 2146). The additional TβRI/TGF-β2 interfaceintroduced by the TβRI/RII hetero-bivalent traps may improve TGF-β2binding to a sufficient level in order to elicit TGF-β2-neutralizationefficacy, not only TGF-β1 and TGF-β3 neutralization. Avidity introducedby hetero-multivalent versions may further accentuate the apparentaffinity and pan-specificity. Similar deductions can be made in the caseof other hetero-valent traps, such as the ActRII/BMPRIa hetero-bivalentand hetero-multivalent traps.

The present design of hetero-valent traps can facilitate tissuepenetration, thereby increasing access to sites of interest. The presentdesign can also provide a shorter half life in systemic circulation,which can be desirable for certain applications such as imaging andother diagnostic applications, as well as where ongoing abundantsystemic distribution of the antagonist is not desirable. In addition,the present design permits linkage of other cargo molecules (for exampleimaging agents like fluorescent molecules), toxins, etc.

For example, and without wishing to be limiting in any manner, thegeneral Structure I

(<bd1>-linker1)_(k)-[{<bd1>-(linker2-<bd2>)-linker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h)

can be modified to add one or more cargo and/or accessory molecules(referred to collectively herein by R₁, R₂, R₃, R₄, etc.), to provideStructure V:

Where bd1, bd2, bd3, bd4, linker1, linker2, linker3, linker4, k, f, n,m, d, and h are defined as in Structure I.

Without limiting the generality of R substituents available, R₁, R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉, may or may not be present; when present, theymay be the same or different, and may independently be one or more of:

-   -   a fusion protein for targeting, for example, but not limited to        such as an antibody fragment (e.g. single chain Fv) and/or a        single domain antibody (sdAb);    -   a radiotherapy and/or imaging agent, for example, but not        limited to a radionuceotide (e.g. ¹²³I, ¹¹¹In, ¹⁸F, ⁶⁴C, ⁶⁸Y,        ¹²⁴I, ¹³¹I, ⁹⁰Y, ¹⁷⁷Lu, ⁵⁷Cu, ²¹³Bi, ²¹¹At), a fluorescent dye        (e.g. Alexa Fluor, Cy dye) and/or a fluorescent protein tag        (e.g. GFP, DsRed);    -   a cytotoxic agent for chemotherapy, for example, but not limited        to doxorubicin, calicheamicin, a maytansinoid derivatives (e.g.        DM1, DM4), a toxin (eg. truncated Pseudomonas endotoxin A,        diphteria toxin);    -   a nanoparticle-based carrier, for example, but not limited to        polyethylene glycol (PEG), a polymer-conjugated to drug,        nanocarrier or imaging agent (e.g. of a polymer        N-(2-hydorxylpropyl)methacrylamide (HPMA), glutamic acid, PEG,        dextran);    -   a drug (for example, but not limited to doxorubicin,        camptothecin, paclitaxel, palatinate);    -   a nanocarrier, for example, but not limited to a nanoshell or        liposome;    -   an imaging agent, for example, but not limited to Supermagnetic        Iron Oxide (SPIO);    -   a dendrimer; and/or    -   a solid support for use in ligand purification, concentration or        sequestration (e.g. nanoparticles, inert resins, suitable silica        supports).

In general, it will not be preferable to have cargo or accessorymolecules in all possible positions, as this may cause steric orelectrostatic complications. However, the effects of adding a cargo oraccessory molecule to any given position or positions on the structurecan be determined routinely in light of the disclosure herein bymodeling the linker between the binding domains and carrying outmolecular dynamics simulations to substantially minimize molecularmechanics energy and reduce steric and electrostatic incompatibilitybetween the linker and the member of the TGF-β superfamily as taughtherein.

It will frequently be preferable to add the cargo or accessory moleculeto the linker portion of the agent, rather to the binding domain, toreduce the likelihood of interference in binding function. However,addition to the binding domain is possible and could be desirable insome instances and the effect of such an addition can be determinedroutinely in advance by modeling the binding agent and the linker withthe proposed addition as described herein.

In certain embodiments of conjugation to cargo molecules and accessorymolecules, the following structures will be produced:

R-[bd]-(linker-[bd])_(n)

[bd]-(R-linker-[bd])_(n)

R-[bd]-(linker-[bd]-R)_(n)

R-[bd]-(R-linker-[bd])_(n)

[bd]-(R-linker-[bd]-R)_(n)

R-[bd]-(R-linker-[bd]-R)_(n)

Conjugation methodologies are somewhat diverse, but typically can beperformed using commercial kits that enable conjugation via commonreactive groups such as primary amines, succinimidyl (NHS) esters andsulfhydral-reactive groups. Some non-limiting examples are: Alexa Fluor488 protein labeling kit (Molecular Probes, Invitrogen detectiontechnologies) and PEGylation kits (Pierce Biotechnology Inc.).

In some instances, the polypeptide may be designed to bindsimultaneously to equivalent but spatially distinct sites on amultimeric ligand. As used herein “multimeric” includes dimeric,trimeric, and greater numbers of units, and “multivalent” includesbivalent, trivalent, and greater numbers of binding domains.

Polypeptides of the invention can be useful as therapeutic agents thatneutralize the action of disease-associated covalently-stabilizeddimeric ligands such as growth factors. They may also have commercialpotential for use as diagnostic agents to detect the presence ofdisease-associated covalently-stabilized dimeric ligands such as growthfactors in imaging and non-imaging diagnostic applications. They canalso be useful in the purification and/or concentration or segregationof ligand in vitro.

The invention also provides a method of designing a hetero-multivalentbinding agent useful in modulating responsiveness of a cell to a memberof the TGF-β superfamily, said method comprising:

-   a) identifying a member of the TGF-β superfamily of interest;-   b) obtaining at least two different polypeptide binding domains    having affinity for different sites on the same member or for    different members of the TGF-β superfamily;-   c) obtaining an unstructured polypeptide linker of at least a number    of amino acids equal to (X/2.5) where X equals the shortest linear    distance between:    -   (i) the C-terminus of an isolated form of the binding domain        that is located at the N-terminus of the linker and that is        specifically bound to its ligand; and,    -   (ii) the N-terminus of an isolated form of the binding domain        that is located at the C-terminus of the linker and that is        specifically bound to its ligand; and,-   d) modelling the linker between the binding domains and carrying out    molecular mechanics and/or dynamics simulations to substantially    minimize the interaction energy and reduce steric and electrostatic    incompatibility between the linker and the member of the TGF-β    superfamily.

The design method can optionally be expanded to further include a stepe) of producing a fusion protein comprising the two polypeptide bindingdomains joined by the unstructured polypeptide linker.

The present invention also encompasses a nucleotide sequence encoding asingle-chain protein produced according to the teachings herein can becloned and inserted into any suitable vector and therefore is veryamenable to production (i.e. there is no requirement for two vectors, orone vector with two promoters, to express two receptor ectodomains).

Large scale production of the hetero-valent ligand traps is anattainable goal, as high yields of 30 mg of purified protein in 500 mlin 293 cells have been obtained with similar other bivalent traps.

In some instances, it may be desirable to permit a computer or othermachine capable of calculation to determine linker length according tothe disclosure herein. Thus, in an embodiment of the invention there isprovided a data storage medium comprising instructions for determiningthe minimum linker length. In an embodiment of the invention there isprovided a data storage medium comprising a means for identifyingacceptable minimal linker length.

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposes only and should not be used to limit the scope ofthe present invention in any manner.

Example 1 Design Strategy of Single-Chain Traps for TGF-β-Family Ligands

1. Single-chain recombinant traps were designed against growth factorsthat belong to the transforming growth factor TGF-β superfamily ofcysteine-knot cytokines according to SCOP (Andreeva et al., 2008, Nucl.Acid Res. 36: D419) and Pfam (Finn et al., 2006, Nucl Acid Res. 34:D247) structural classifications. More specifically, these growthfactors including, for example, TGF-βs, activins and BMPs, share thesame 3D architecture and form covalent disulfide-linked homodimers. Themethod disclosed herein is applicable to all members of theTGF-βsuperfamily, including TGF-β1, -β2, -β3; activin βA, βB, βC, βE;bone morphogenetic proteins (BMP) 2-15; growth differentiation factors(GDF) 1, 3, 8 (myostatin), 9 and 15; Nodal; Inhibin α; anti-Mullerianhormone (AMH); Lefty 1 and 2; Arteman, Persephin and Neurturin.

2. Single-chain recombinant traps against TGF-β superfamilygrowth-factors were designed from the extracellular portion of theircognate natural receptors. The extracellular segment of all these TGF-βsuperfamily receptors contain a single structured domain that belongs tothe snake-toxin family according to SCOP (Andreeva at al., 2008, Nucl.Acid Res. 36: D419) and Pfam (Finn et al., 2006, Nucl Acid Res. 34:D247) structural classifications. The complete extracellular portion ofthese receptors typically includes unstructured segments flanking theirfolded ligand-binding domain. These unstructured extracellular portionswere apparent from the experimentally determined 3D structures availablefrom the PDB database (Berman et al., 2000, Nucl. Acid Res. 28: 235),e.g., crystal structures for type II TGF-β receptor ectodomain (Hart etal., 2002 Nat. Struct. Biol. 9: 203; Boesen et al., 2002, Structure 10:913; Groppe et al., 2008, Mol. Cell 29: 157), type I TGF-β receptorectodomain (Groppe et al., 2008, Mol. Cell 29:157), type Ila activinreceptor ectodomain (Allendorph et al., 2006, Proc. Natl. Acad. Sci. USA103: 7643), type IIb activin receptor ectodomain (Thompson at al., 2003,EMBO J. 22: 1555; Greenwald at al., 2004, Mol. Cell 15: 485), type I BMPreceptor ectodomain (Kirsch et al., 2000, Nat. Struct. Biol. 7: 492), orthe NMR structure of the type II TGF-β receptor ectodomain (Deep et al.,2003, Biochemistry 42: 10126)]. In the absence of experimental data, asfor example in the case the extracellular region of the IIb splicingvariant of the TGF-β type II receptor, unstructured extracellularsegments were defined by: (i) sequence portions falling outside of thefolded ligand-binding domain boundaries located by comparative analysisagainst structurally characterized homologs, and (ii) predictions basedon knowledge-based algorithms, e.g., DISOPRED (Ward et al., 2004, J.Mol. Biol. 337: 635). Amino acid sequences corresponding to theunstructured (i.e., flexible) and structured (i.e., folded,ligand-binding domain) regions from the ectodomains of several receptorsof TGF-β-superfamily growth factors, are given in FIGS. 1A and 1B,respectively.

3. Homo-bivalent single-chain recombinant traps (TβR-II)², (TβR-IIb)²,(ActR-IIb)² and (BMPR-Ia)² have previously been designed, produced, andtested as described in published PCT application WO 2008/113185.

4. Heterovalent single-chain recombinant traps against TGF-β-superfamilygrowth factors disclosed herein were designed similarly with thehomovalent single-chain traps previously disclosed (WO/2008/113185,incorporated herein by reference), based on the experimentallydetermined binding mode between TGF-β-family ligands and theextracellular portion of their cognate natural receptors. Theligand-receptor binding mode was provided at atomic level by thehigh-resolution 3D structures available for several members of theTGF-β-superfamily ligands in complex with their cognate receptorectodomains. Specifically, ternary ligand-receptor assemblies between aparticular TGF-β-superfamily growth factor and ectodomains fromdifferent receptor types have been determined for the TGF-β3, TβR-II-EDand TβR-I-ED complex (Groppe at al., 2008, Mol. Cell 29:157) and for theBMP-2, ActR-IIa-ED and BMPR-Ia-ED complex (Allendorph et al., 2006,Proc. Natl. Acad. Sci. USA 103: 7643). These structures provide therelative spatial orientation between four separate receptor ectodomainchains (molecules) binding simultaneously onto one covalentlyhomodimerized ligand molecule, i.e., 2:2:1high-affinity-receptonlow-affinity-receptorligand stoichiometry. Suchstructures were used as guides to design hetero-bivalent,hetero-trivalent and hetero-tetravalent single-chain traps ofTGF-β-superfamily growth factors and are useful in designingsingle-chain traps for other suitable ligands of interest involving theTGF-β superfamily.

5. Hetero-bivalent and hetero-multivalent single-chain traps ofTGF-β-family ligands were designed as unnatural fusion proteinsconsisting of the sequence (excluding the signal peptide) of the naturalextracellular portion of one receptor repeated one or more times and thesequence (excluding the signal peptide) of the natural extracellularportion of another receptor repeated one or more times. FIG. 4 describesheterovalent single-chain traps with natural linkers for TGF-β ligands,where structured and unstructured regions are based on experimental dataas presented in FIGS. 1A and 1B. This design resulted in constructs withtwo or more structured domains for binding to select TGF-β-superfamilyligand(s), spaced by unstructured flexible linker(s) formed by fusingthe unstructured C-terminus of one domain to the unstructured N-terminusof another domain. The natural linkers can also be progressivelysubstituted by artificial sequences as well as varied in length (FIGS.5, 6). Hetero-multivalent designs result from appropriate assemblies ofhomo-bivalent and hetero-bivalent designs. From thermodynamic andkinetic considerations, it was expected that multivalent receptorectodomains would provide increased ligand-binding affinities and slowerligand-dissociation rates relative to single-domain receptorectodomains. In the specific case of heterovalent traps directed againstTGF-β isoforms, the heterovalent design was also aimed at increasing thespecificity spectrum to include all TGF-β isoforms, i.e., TGF-β2, notonly TGF-β1 and TGF-β3.

Example 2 Feasibility Assessment Procedure for Designed Single-ChainBivalent Traps

To the extent to which the structures of various TGF-β-superfamilygrowth factors are conserved, the structures of their cognate receptorectodomains are conserved, and the 2:1 receptor-ligand bindingstoichiometry is conserved, the concept of fusing two natural receptorectodomain sequences to produce single-chain hetero-bivalent traps withimproved in vitro ligand binding affinity and ligand neutralizingactivity relative to respective monovalent receptor ectodomains isapplicable to the entire family of TGF-β ligands. The feasibility ofthese ligand traps can be theoretically assessed routinely by followingthe stepwise procedure outlined below. Although the procedure ispresented for hetero-bivalent single-chain traps, it also applies toother designs, e.g., hetero-multivalent single-chain traps (disclosedherein).

1. The linear distance is measured between the C-terminal main-chaincarbon atom of one domain and the N-terminal main-chain nitrogen atom ofthe other domain when bound to the covalently-dimerized ligand.Alternate structures of the complex reflecting internal geometricalflexibility in the homodimerization mode of the disulfide-stabilizedligand when bound to the receptor ectodomains can be included in thedesign process. A computer hardware equipped with commercial/publicsoftware appropriate for manipulating molecular structures on anavailable graphics device can be routinely employed to this end.

2. The linear distance (in Å units, 1 Å=10⁻¹⁰ m) is divided by a factorof 2.5 to calculate the minimum number of amino acid residues that theflexible linker should posses (Table 3) in order to allow simultaneousbinding of the folded domains to their binding sites on the homodimericligand. The 2.5 factor is based on the Cα-Cq extent of fully extendedlinkers, which peaks at 3.0 Å (George and Hering a, 2002, Protein Eng.15: 871), minus an average tolerance of 0.5 Å per amino acid residue toallow for deviations of the linker path from linearity.

TABLE 3 Linker characteristics for select examples of hetero-bivalentsingle-chain traps of TGF-β-family growth factors. Reference ResiduesLinear Minimum Receptor structures in distance residues Targetedectodomain (PDB “natural” (Å) for required for Single-chain trapligand(s) used entries) linker linkage linkage^((a)) TβR-I/II-v1 TGF-β1TβR-I-ED, 2PJY 7 14 6 TGF-β2 TβR-II-ED TGF-β3 TβR-I/II-v1a TGF-β1TβR-I-ED, 2PJY 8 14 6 TGF-β2 TβR-II-ED TGF-β3 TβR-I/II-v1b TGF-β1TβR-I-ED, 2PJY 9 14 6 TGF-β2 TβR-II-ED TGF-β3 ActR-IIa/BMPR-Ia-v1 BMP-2ActR-IIa- 2GOO 24 55 22 ED, BMPR- Ia ActR-IIa/BMPR-Ia-v1a BMP-2ActR-IIa- 2GOO 26 55 22 ED, BMPR- Ia ActR-IIa/BMPR-Ia-v1b BMP-2ActR-IIa- 2GOO 33 55 22 ED, BMPR- Ia ^((a))Minimum number of residuesrequired for linkage represents the structure-based linear distance forlinkage (Å) divided by a factor of 2.5.

3. The number of amino acid residues in the unstructured linker portionof the hetero-bivalent single-chain trap should be at least equal to theestimated minimum number of linker residues required. Receptor isoformsthat differ in the length of the extracellular unstructured segments,such as the TGF-β receptor isoforms II and IIb (FIG. 2A), can beincluded in the design process. The natural sequence-based linker canalso be shortened up to the estimated minimum number of amino acidresidues without significantly impairing the ligand binding affinity andneutralizing activity of the trap. A preferable location for shorteningthe unstructured linker is from the point of in either or bothdirections relative to the amino acid sequence, as exemplified byTbR-I/II-v1, -v1a, -v1b constructs for TGF-b traps andActR-IIa/BMPR-Ia-v1, -v1a, -v1b constructs for BMP-2 traps (Table 3,FIG. 5). Example of shortened natural linkers that can be utilized insingle-chain trap design are given in FIG. 2C.

As listed in Table 3, the required minimal length of the linker variesbetween various single-chain traps of TGF-β-superfamily growth factors.An upper limit for the length of the unstructured linker is not defined.Hence, ligand binding agent (trap) constructs with linkers comprisingunstructured sequence segments repeated in whole or in part areenvisioned to comply with bivalent design and preserve the desiredcharacteristics of the trap. The natural linker can be progressivelysubstituted by artificial sequences, which may or may not result indifferent linker lengths. Examples of linkers longer than the naturallinker designed by repeating of natural sequence or by introducing ofartificial sequence are given in FIG. 2D. Examples of introducing ofartificial sequences as linkers in the design of hetero-bivalent trapsare given in FIG. 6.

4. Finally, atomic-level theoretical analysis is to be carried out,where the linker is modeled between the structured domains and themolecular structure of the trap-ligand complex is refined by minimizingthe molecular mechanics energy and by carrying out molecular dynamicssimulations (Cornell et al., 1995, J. Am. Chem. Soc. 117: 5179). Thismay, in some cases, highlight regions of steric and/or electrostaticincompatibility between the trap's linker and the growth-factor, andsuggest that the length and/or composition of the linker may beincompatible with the bivalent design, even if the linker complies withthe minimum number of amino acids requirement as per step (3.) above. Ifthe linker can be accommodated without affecting the simultaneousbinding of the structured domains to their binding sites on the ligand,then the trap construct is deemed feasible for the proposed application.Computer hardware equipped with commercial/public software appropriatefor manipulating molecular structures on an available graphics device,and for performing energy calculation and simulation based on molecularmechanics force fields, e.g., the AMBER force field (Cornell et al.,1995, J. Am. Chem. Soc. 117: 5179), can be routinely employed by oneskilled in the art in order to carry out this structural modelinganalysis. Examples of molecular mechanics energy-refined models of twosingle-chain hetero-bivalent traps, TβR-I/II-v1 and ActR-IIa/BMPR-Ia-v1,bound to their respective growth factors are shown in FIG. 7. Theseatomic-level models indicate the steric and electrostatic compatibilityof the designed linker in the trap-ligand complex. These models alsorepresent starting points for further computer-based optimization oflinker composition and length. More detailed atomic-level solutionstructure based on molecular dynamics simulations ca be carried outroutinely to further characterize the binding mode of these and otherconstructs, as exemplified for the homo-bivalent traps disclosedpreviously (WO/2008/113185).

Example 3 Demonstration that Shale-Chain Homobivalent Traps (TβRII)² and(TβRIIb)2 are Potent Neutralizers of TGF-β1 and TGF-β3 but not TGF-β2

Homo-bivalent single-chain recombinant traps (TβRII)² and (TβRIIb)² wereprepared as previously described in published PCT application WO2008/113185. The ability of purified (TβRII)² to neutralize TGF-β wastested on Mv1Lu cells having a TGF-β-responsive luciferase reporter geneand compared with TβRII-ED monomer, TβRII-Fc, and pan-specific TGF-βneutralizing antibody 1 D11 (FIG. 3). The resulting inhibition curves(FIGS. 3A and 3B, and data not shown) allowed determination of theaverage IC₅₀, a measure of neutralization potency for each TGF-β isoform(summarized in Table 4). (TβRII)² and (TβRIIb)² traps were respectively˜100-fold and 1000-fold more potent than TβRII-ED for neutralizingTGF-β1 and TGF-β3. However, these homo-bivalent traps were unable toneutralize TGF-β2.

TABLE 4 Trap IC₅₀s (nM) determined from TGF-β neutralization curves.Trap IC50 for TGF-β1 IC50 for TGF-β2 IC50 for TGF-β3 (TβRII)2   1.359(0.459, n = 3) ^(a)) No neutralization 0.336 (0.125, n = 5) (TβRIIb)20.098 (0.021, n = 4) No neutralization 0.045 (0.012, n = 3) TβRII-Fc0.506 (0.506, n = 4) No neutralization 0.323 (0.067, n = 3)TβRII-ED >100 No neutralization >100 1D11 antibody 1.429 (0.676, n = 4)8.674 (0.303, n = 2) 0.029 (0.022, n = 2) ^(a)) SEM for n experiments,each performed with triplicate samples.

Example 4 Examples of Hetero-Bivalent TβRI/RII Traps thatPan-Specifically Neutralize TGF-β

In order to assess the functionality of heterovalent single-chain traps,two hetero-bivalent trap versions depicted in FIGS. 4A and 4B, namelyTβR-I/II-v1 and TβR-I/II-v2, were transiently expressed in HEK 293cells. Conditioned media containing the secreted trap was then seriallydiluted and tested for neutralization of TGF-β1 or TGF-β2 using Mv1Luluciferase reporter cells as a readout (FIGS. 8A and B). Both trapversions neutralized TGF-β1 and TGF-β2.

Construction and Cloning of Hetero-Valent TβRI/RII Trans

TβR-I/II-v1 and TβR-I/II-v2 constructs (shown in FIGS. 4A and 4B) wereassembled by PCR using appropriate primers and human TβRI and TβRIItemplate sequences and subsequently cloned into mammalian expressionvector pTT2 (Durocher et al., 2002, Nucleic Acids Res. 30: E9) fortransient expression in HEK293 cells or cloned into lentivirusexpression vector Tet07CSII-CRS-mcs (Broussau et al, 2008, Mol. Ther.16: 500) for transduction and stable expression in CHO cells. Eachconstruct was preceded by the following VEGFsignal sequence/Histag/Thrombin cleavage site:

[MNFLLSWVHWSLALLLYLHHAKWSQA]APMAEGGGQNHHHHHHHHGGSFNPR. (SEQ ID NO: 108)

Small-Scale Transient Transfections:

Modified human embryonic kidney cells (293-EBNA1 clone 6E) stablyexpressing EBNA1 were transfected using 25 kDa linear polyethylenimine(PEI) (Poysciences, Warrington, Pa.) as described below (and Durocher etal., 2002, Nucl. Acid Res. 30: e9)). The cells growing as suspensioncultures in Freestyle medium (Invitrogen) were transfected at 1×10⁶cells/ml with a fixed amount of pTT2-trap plasmid DNA and 2 ug/ml PEI,as follows: Five hundred microliters of the suspension culture wasdistributed per well in a 12-well plate. DNA was diluted in Freestylemedium (in a volume equivalent to one-tenth of the culture to betransfected), PEI was added, and the mixture immediately vortexed andincubated for 10 min at room temperature prior to its addition to thecells. Following 3 h incubation with DNA-PEI complexes, culture mediumwas completed to 1 ml. The culture was harvested 5 days aftertransfection and the media was clarified by centrifugation at 3500 g for10 min and sterile filtered. Aliquots of conditioned media were analyzedfor TGF-β neutralizing activity.

Comparison of the Antagonistic/Inhibitor Potencies of Various BindingAgents by Mv1Lu Luciferase Reporter Assays

Mink lung epithelial cells, stably transfected with the TGF-β-responsivePAI-1 promoter fused to the firefly luciferase reporter gene (Abe etal., 1994, Anal. Biochem. 216: 276), were used. These cells were platedin 96-well tissue culture plates (2×10⁴ cells/well) in Dulbecco'smodified Eagle's medium containing 5% fetal bovine serum and wereallowed to attach for at least 6 h at 37° C. Cells were then washed withphosphate buffered saline (PBS), and the medium was replaced byDulbecco's modified Eagle's medium containing 1.0% fetal bovine serumand 0.1% bovine serum albumin (DMEM-1, 0.1% BSA). Various concentrationsof purified single-chain TGF-β trap, TβRII-Fc (R&D Systems), or TGF-βneutralizing antibody 1 D11 (R&D Systems) were mixed with 20 pM TGF-β inDMEM-1, 0.1% BSA and added to the cells. After 16 hr. incubation at 37°C., the medium was removed, and the cells were washed once with PBS.Cells were then lysed with 25 μl reporter lysis buffer (Promega Corp.)and assayed for luciferase activity using the Promega luciferase assaykit according to the manufacturers instructions. Luminescence wasmeasured in a MRX (Dynex Inc.) or Lumioskan RS (Global MedicalInstrumentation, Inc.) microplate reader. The activity is expressed asthe percentage of the maximum TGF-β1 activity (i.e. in the absence ofany antagonist) or relative luciferase units (RLU) (see examples shownin FIGS. 3, 8 and 9).

TβRI/RIIv1 Lentivirus-Transduced CHO Cell Cultures and ProteinPurification:

Transduced CHO cells stably expressing TβR-I/II-v1 trap were grown in 2liter suspension culture. The culture medium was harvested and trapprotein was purified by immobilized metal affinity chromatography onFractogel-Cobalt column as previously described (Cass et al., 2005,Protein Expr. Purl. 40: 77) except that wash and elution steps contained25 mM and 300 mM imidazole respectively. A 10 ml column packed with 5 cmTalon Metal Affinity Resin (BD Biosciences, Mississauga, Ont.) and wasequilibrated with 10 column bed volumes (CVs) of Talon Wash Buffer (TWB:50 mM sodium phosphate, 300 mM NaCI, pH 7). The conditioned medium waspassed through a 0.22 μm filter, and then loaded by gravity. The columnwas washed with 10 CVs of TWB and (TβRII)² was eluted in 1 ml fractionsusing 300 mM imidazole in TWB. Eluted trap protein was then desalted inPBS using a HiPrep 26/10 desalting column (GE-Healthcare) as recommendedby the manufacturer. Protein concentration was determined by Bradfordusing BSA as a standard.

Example 5 Purified TβR-I/II-v1 Neutralizes Both TGF-β1 and TGF-β2

TβR-I/II-v1 was stably expressed in CHO cells; ˜45 mgs trap protein waspurified from a 2 liter culture. Neutralization curves determined forpurified TβR-I/II-v1 indicated IC₅₀s of 0.4 nM and 11.9 nM for TGF-β1and TGF-β2, respectively (FIGS. 9A and 9B). Full neutralization of bothTGF-β isoforms was observed with ˜200 nM trap. This proved thatheterovalency improved trap affinity and potency as compared tohomo-bivalent TGF-β traps for targeting multiple TGF-β isoforms.

The embodiments and examples described herein are illustrative and arenot meant to limit the scope of the invention as claimed. Variations ofthe foregoing embodiments, including alternatives, modifications andequivalents, are intended by the inventors to be encompassed by theclaims. Furthermore, the discussed combination of features might not benecessary for the inventive solution.

All documents, including patents, patent applications, journal articles,etc are incorporated herein by reference in their entirety.

1. A hetero-multivalent binding agent with affinity for one or more thanone member of the TGF-β superfamily, said agent comprising the generalStructure I:(<bd1>-linker1)_(k)-[{<bd1>-linker2-<bd2>-linker3_(f)-}_(n)-(<bd3>)_(m)-(linker4-<bd4>)_(d)]_(h),where: n and h are independently greater than or equal to 1; d, f, m andk are independently equal to or greater than zero; bd1, bd2, bd3 and bd4are polypeptide binding domains independently having an affinity for amember of the TGF-β superfamily, wherein at least two of bd1, bd2, bd3,and bd4 are different from each other; and, linker1, linker2, linker3and linker4 are unstructured polypeptide sequences; wherein the numberof amino acids in each linker is determined independently and is greaterthan or equal to X/2.5, where X equals the shortest linear distancebetween: (a) the C-terminus of an isolated form of the binding domainthat is located at the N-terminus of the linker and that is specificallybound to its ligand; and, (b) the N-terminus of an isolated form of thebinding domain that is located at the C-terminus of the linker and thatis specifically bound to its ligand.
 2. The agent of claim 1 wherein themember of the TGF-β superfamily to which the binding domains haveaffinity is selected from the group consisting of: TGF-β1, TGF-β2,TGF-β3, activin βA, activin βB, activin βC, activin βE, bone morphogenicprotein (BMP) 2, BMP 3, BMP4, BMP 5, BMP 6, BMP 7, BMP 8, BMP 9, BMP 10,BMP 11, BMP 12, BMP 13, BMP 14, BMP 15, growth differentiation factor(GDF) 1, GDF 3, GDF 8, GDF 9, GDF 15, Nodal, Inhibin α, anti-MullerianHormone, Lefty 1, Lefty 2, arteman, Persephin and Neurturin.
 3. Theagent of claim 2 wherein the member of the TGF-β superfamily to whichthe binding domains have affinity is selected from the group consistingof: TGF-β1, TGF-β2, TGF-β3, BMP2, GDF 8, and activin.
 4. The agent ofclaim 1 wherein the linker is between 25 and 60 amino acids in length.5. The agent of claim 1 wherein bd4 is the same as bd1, bd 2 is the sameas bd3, h>0, and d, f, m, and n=1.
 6. The agent of claim 1 comprisingany one or more of SEQ ID No 44 to 64, or a sequence substantiallyidentical thereto.
 7. The agent of claim 1 comprising one or more of SEQID NO 1-11, 18-43, or 65-107 as a linker sequence.
 8. The agent of claim1 wherein one or more of bd1, bd2, bd3, and bd4 is selected from one ofSEQ ID NO 12-17.
 9. The agent of claim 1 having the general structure V:

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, may be present or not, andwhen present, may independently be one or more of a protein fortargeting, a single domain antibody, a radiotherapy agent, an imagingagent, a fluourescent dye, a fluorescent protein tag, a cytotoxic agentfor chemotherapy, a nano particle-based carrier, a polymer-conjugated todrug, nanocarrier or imaging agent, a stabilizing agent, a drug, ananocarrier, a support, and a dendrimer; and wherein the remainingfeatures are as defined in claim
 1. 10. A method of designing ahetero-multivalent linker useful in modulating responsiveness of a cellto a member of the TGF-β superfamily, said method comprising: a)identifying a member of the TGF-β superfamily of interest; b) obtainingat least two different polypeptide binding domains having affinity fordifferent sites on the same member or for different members of the TGF-βsuperfamily; c) obtaining an unstructured polypeptide linker of at leasta number of amino acids equal to (X/2.5), wherein the number of aminoacids in each linker is determined independently and is greater than orequal to X/2.5, where X equals the shortest linear distance between: (i)the C-terminus of an isolated form of the binding domain that is locatedat the N-terminus of the linker and that is specifically bound to itsligand; and, (ii) the N-terminus of an isolated form of the bindingdomain that is located at the C-terminus of the linker and that isspecifically bound to its ligand; and d) modeling the linker having oneof the binding domains covalently attached at each end, and carrying outmolecular mechanics and/or dynamics simulations to substantiallyminimize the interaction energy and reduce steric and electrostaticincompatibility between the linker and the member of the TGF-βsuperfamily.
 11. The method of claim 10 further including a step e)producing a fusion protein comprising the two polypeptide bindingdomains joined by the unstructured polypeptide linker.
 12. A nucleicacid sequence encoding a hetero-multivalent binding agent of claim 1.13. A method of modulating the response of a cell to a TGF-β superfamilymember in its environment, said method comprising exposing the cell toan agent of claim
 1. 14. Use of an agent of claim 1 to purify ligand orconcentrate ligand in a sample.
 15. (canceled)
 16. Use of an agent ofclaim 9 in diagnosis of a condition characterized in whole or part by anabnormality in levels of one or more TGF-β superfamily members in thebody or a portion thereof.
 17. Use of an agent of claim 9 in targetingdelivery of a compound to a site of interest within a body.